Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs’ genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.


De novo genome assembly and annotation
The whole genome sequencing of a 25-year-old female AKV (RV4F) from Nakhon Ratchasima Zoo was conducted using both PacBio long-read and MGISEQ-2000 short-read platforms.Additionally, the ORG.one project (https:// nanop orete ch.com/ oo) supported two MinION flow cells for Nanopore long-read sequencing.Details on genome assembly, GC content, and Benchmarking Universal Single-Copy Orthologs (BUSCO) assembly scores for two approaches -the PacBio and MGI hybrid sequencing with Purged analysis (PacBio-MGI-Purged), and the Nanopore and MGI hybrid sequencing (Nanopore-MGI) -are presented in Table 1.Due to insufficient Nanopore sequencing data (10 × coverage, total bases 13.5 Gb) for genome assembly, we assembled the reference genome using raw reads sequenced by PacBio (1228 × coverage, total bases 1548.3Gb) and MGISEQ-2000 only (109 × coverage, total bases 140.6 Gb).Ultimately, we generated a total genome length of 1.29 Gb (1230 × coverage) for the AKV (GenBank accession number: PRJNA827941), with a scaffold N50 of 28.73 Mb, after removing low-quality and duplicated reads.The completeness of the AKV genome was evaluated using 8,338 single-copy orthologs.It achieved 97.4% completeness with 1.1% complete and duplicated BUSCOs, 0.5% corresponding to fragmented BUSCOs and 2.1% indicating missing BUSCOs.Additional details, such as repeat elements, total protein-coding genes, non-coding RNAs, KEGG pathways and Gene Ontology (GO) categories of putative genes for the AKV assembly, can be found in Supplementary Tables 1 and 2 and Fig. 1.We annotated a total of 19,388 protein-coding genes, covering a gene length totality of 236 Mb with an average gene size of 12,148 nucleotides.Out of the annotated genes, 17,444 (89.97%) were functionally annotated through GO, and 13,656 (70.44%) were functionally annotated through KEGG.Exons had a combined length of 31.47Mb, with an average length of 185 bp, while introns had a total length of 204.07 Mb, with an average length of 1352 bp.The average number of exons per gene was 8.79 and the average number of introns per gene was 7.79.The GC contents in exons and introns were 52.08 and 42.67%, respectively.Sequences with the annotation of endogenous retrovirus were counted (Supplementary Table 3).The proportions of the total endogenous retrovirus sequences in the AKV genome were discovered to be 33%, 16% and 51% for the Env protein, Gag protein and Pol protein, respectively.

Phylogenetic analyses based on the nuclear genome
To illustrate the genomic relatedness among species, a phylogenetic tree based on the nuclear genome was constructed using amino acid sequences of 1259 single-copy orthologous genes from 13 species.These species included 10 birds in the order Accipitriformes: white-tailed eagle (Haliaeetus albicilla), bald eagle (Haliaeetus leucocephalus), northern goshawk (Accipiter gentilis), harpy eagle (Harpia harpyja), black hawk-eagle (Spizaetus tyrannus), golden eagle (Aquila chrysaetos), Asian king vulture (S. calvus), black-chested snake eagle (Circaetus pectoralis), osprey (Pandion haliaetus) and secretary bird (Sagittarius serpentarius).Additionally, two NWV species, California condor (Gymnogyps californianus) and turkey vulture (Cathartes aura), were also included, with red junglefowl (Gallus gallus) serving as the outgroup (Fig. 2).In addition, the estimated divergence time between AKV and other Accipitrid hawks and eagles within the same family was approximately 13.13 million years ago (Mya), while the estimated divergence time between AKV and the NWV was approximately 38.37 Mya.

Species-specific orthologous gene families
Species-specific gene families in the genomes of three vulture species, the AKV, California condor and turkey vulture, were analyzed using OrthoVenn2 21 .Among a total of 14,214 families, 5183 were orthologous families being shared between two species, while 703 gene families were unique to a single species.Among the 242 AKVspecific gene families, approximately 65% (158 families) could be annotated using the UniProtKB/Swiss-Prot database.GO enrichment analysis revealed major families for Gag-Pol polyprotein, associated with viral genome integration to host DNA (GO:0044826), families for envelope glycoprotein, involved in virion attachment to the host cell (GO:0019062), and families for E3 ubiquitin-protein ligase ICP0, associated with the suppression of host interferon regulatory factor 7 (IRF7) activity (GO:0039557) (Fig. 3a, Supplementary Table 4).For the NWVs, 457 gene families were specific to this group.Out of 430 annotated gene families, 358 were associated with biological processes, 46 with molecular functions and 26 with cellular components.The primarily enriched specific gene families of NWVs included families for olfactory receptor families, associated with smell perception (GO:0007608, GO:0004984) (Fig. 3a, Supplementary Table 4).
Notably, species-specific genes in the AKV genome were also compared within the Accipitridae clade, which includes the white-tailed eagle, northern goshawk, harpy eagle, golden eagle and black-chested snake eagle.Out of 22,952 families, 20,001 orthologous families were found in at least two species with 2951 observed as singlecopy gene families.A total of 173 AKV-specific gene families were represented in a Venn diagram (Fig. 3b).Among these, 78 gene families could be annotated using the SWISS-PROT database.Similar to the results for species-specific gene families among the three vulture species, we observed gene families for envelope glycoprotein, which is related to virion attachment to host cell (GO:0019062).Additionally, we found other families corresponding to retrovirus integration including families for Gag-Pol polyprotein associated with virion assembly (GO:0019068), families for E3 ubiquitin-protein ligase IE61 involved in the modulation of host protein ubiquitination (GO:0039648), families for Pro-Pol polyprotein associated with viral penetration into the host nucleus (GO:0075732), families for Gag-Pol polyprotein associated with DNA recombination (GO:0006310), and families for Gag polyprotein associated with viral budding via the host ESCRT complex (GO:0039702) (Supplementary Table 5).www.nature.com/scientificreports/

Positively selected genes
Thirteen avian genomes were analyzed for PSGs, including 12 from Accipitriformes and one from Galliformes.
The results revealed the presence of 47 significant PSGs in AKV.Several genes with single amino acid substitutions were identified, notably, baculoviral IAP repeat containing 5 (BIRC5), CD274, aryl hydrocarbon receptor (AHR), aggrecan (ACAN), myoferlin (MYOF), actinin alpha 4 (ACTN4), mannose receptor C-type 1 (MRC1) and spermidine synthase (SRM).Predictions suggest that these substitutions may negatively impact the genome when compared to the red junglefowl reference sequence.The PSG families associated with these genes revealed significant enrichment in various biological processes.Notably, the positive regulation of the cell cycle checkpoint (GO:1901978) showed the highest fold enrichment, with the BIRC5 gene displaying a fold enrichment exceeding 50.Additionally, the response to tumor cells (GO:0002834) featured CD274 and AHR genes with a fold enrichment surpassing 40.The third-ranked process implicated the regulation of stem cell proliferation (GO:0072091), with ACAN exhibiting a fold enrichment of 10.53, while muscle cell development (GO:0055001), containing MYOF and ACTN4, demonstrated a fold enrichment of 9.73.Furthermore, cellular response to cytokine stimulus (GO:0071345) was observed, involving CD274, MRC1, ACTN4 and SRM genes, with a fold enrichment of approximately 3. The major molecular functions were actin binding (GO:0003779), associated with the ACTN4 gene, and protein dimerization activity, which encompassed the ACTN4, BIRC5, AHR and SRM genes.(Supplementary Tables 7, 8).

SNP and genotype calling
RADseq was performed on whole blood samples from five AKVs.The raw reads were aligned to our de novo AKV assembly, resulting in 626,584 SNPs.SNPs on Z and W chromosomes were filtered out after aligning against the Z and W chromosomes of the golden eagle (Accession No GCF_900496995.4) to remove low quality SNPs and eliminate potential bias from SNPs on putative sex chromosomes.After filtering, 52,167 SNPs remained.Subsequently, two SNP datasets were prepared, including a non-linkage disequilibrium (LD)-pruned dataset and an LD-pruned dataset.The non-pruned LD dataset, containing 52,167 SNPs, was used for detecting runs

Population structure and genetic relatedness based on identity by descent
In the context of phylogenetic analysis, AKV2M and AKV3M were found to form a sister group, as did AKV4F and AKV5F.AKV1M, AKV2M and AKV3M exhibited a closer genetic similarity with shorter branch lengths www.nature.com/scientificreports/when compared to AKV4F and AKV5F, implying that AKV1M, AKV2M and AKV3M likely belong to the same subpopulation (Fig. 5a).The MDS plot illustrated three potential clusters for those five AKV samples.The initial cluster comprised AKV1M, AKV2M and AKV3M; the second cluster consisted of AKV4F, while the third cluster encompassed AKV5F (Fig. 5b).Additionally, the IBD analysis, as depicted in the heatmap (Fig. 5c), indicated that all five AKV individuals were genetically unrelated.

Inbreeding evaluation
The inbreeding coefficients computed from the ROH length (F ROH ) for the five AKV individuals were as follows: AKV1M = 0.09, AKV2M = 0.08, AKV3M = 0.07, AKV4F = 0.17 and AKV5F = 0.15.Their average F ROH was 0.11 ± 0.05.In addition, we calculated the inbreeding coefficient based on excess homozygosity (F HOM ) using PLINK.The genetic diversity indices for each individual AKV, including observed and expected homozygosity and F HOM , are summarized in Table 2.We observed a positive correlation between F ROH and F HOM (Pearson's correlation test, correlation coefficient = 0.98, p < 0.01).The relationship between the ROH count and the ROH length is illustrated in Fig. 6.Notably, AKV4F and AKV5F exhibited the longest ROH lengths, exceeding 16 Mb.

Discussion
This study presented the first reference genome of AKV and conducted comparative genomic analyses with published genomes of NWVs, including California condor and turkey vulture, as well as other bird species within the Accipitridae family.Additionally, the RADseq-based genome-wide SNPs were utilized to elucidate the genetic relatedness among five AKVs in Thailand.The AKV reference genome assembly was constructed from raw reads obtained through a combined PacBio-MGI-Purged method.Purge_dups analysis was employed to identify and eliminate duplicate regions, haplotigs and heterozygous overlaps, resulting in enhanced assembly continuity while preserving completeness.Although the MinION portable sequencer theoretically provides up to 20 Gb of data 24 , in the present study, 13.5 Gb of raw reads were obtained from two flow cells, generously supported by ORG.one.Given the AKV genome size of 1.29 Gb, at least six flow cells would be required to achieve 30 × coverage.Consequently, the output was insufficient for a whole-genome assembly of the AKV.Nonetheless, the AKV genome assembly achieved a scaffold N50 of 28.73 Mb and a genome size of 1.29 Gb, surpassing those of the NWVs.Specifically, the California condor has a scaffold N50 value of 17.1 Mb and a genome size of 1.24 Gb, whereas the turkey vulture has a scaffold N50 value of 15.2 Kb and a genome size of 1.15 Gb 25,26 .Moreover, when compared to closely related taxa with scaffold N50 values ranging from 0.057 to 58.1 Mb and genome sizes ranging from 1.18 to 1.27 Gb, the AKV genome size falls within this range of scaffold N50 lengths.The completeness of the AKV genome, at 97.4%, is comparable to that of sister taxa (white-tailed eagle, bald eagle, northern goshawk, harpy eagle, black-hawk eagle and golden eagle), which exhibit genome completeness ranging from 91.2 to 99.2% [26][27][28] .It is noteworthy that different sequencing techniques may impact results, as most of the published reference genomes mentioned above were generated using Illumina short-read sequencing, except for the genomes of the California condor, harpy eagle, golden eagle and northern goshawk, which were sequenced using both Illumina short-read and PacBio long-read sequencing.Additionally, genomes with larger scaffold N50 values were assembled at the chromosome level 27,28 .The AKV genome assembly revealed that repetitive elements constitute 7.8% of the genome.Repetitive elements in avian genomes typically range from 4 to 10% of the genome assembly, except for the downy woodpecker, which contains repetitive elements accounting for 22% of the genome assembly due to species-specific LINE expansions 29 .Using a combination of short-read and long-read sequencing, along with advanced bioinformatic tools, could potentially enhance the annotation of repetitive elements 30 .
In this study, a total of 19,388 predicted protein-coding genes were identified in the AKV.When compared to other species available in the NCBI database, the turkey vulture (accession number: GCA_000699945.1) and the California condor (accession number: GCA_018139145.2) were found to have 11,361 and 15,144 protein-coding genes, respectively.Conversely, species more closely related to AKV, such as the golden eagle (accession number: GCA_900496995.4) and bald eagle (accession number: GCA_000737465.1), were annotated with 16,702 and 15,212 protein-coding genes, respectively.It is worth noting that gene annotation using RNA sequencing-based www.nature.com/scientificreports/methods likely contributed to improved annotations 31 .Additionally, divergence time estimation was conducted based on 1259 single-copy orthologous genes between AKV and other species.The estimated divergence time between OWVs and NWVs was determined to be 38.37 Mya, while the estimated divergence time between www.nature.com/scientificreports/AKV and sister taxa, including the white-tailed eagle, bald eagle, northern goshawk, harpy eagle, black hawkeagle and golden eagle, was estimated to be 13.13Mya.These estimations differ from previous studies, where the estimated divergence time between OWVs and NWVs was 60 Mya, and between cinereous vultures and bald eagles, it was 18 Mya 15 .These variations may arise from differences in the methods and calibration points utilized in the analysis.
In our comparative genomic analysis, the investigation of species-specific gene families revealed enriched gene families associated with virus infection.These functions included virion attachment to host cells, viral genome integration, virion assembly and budding.Endogenous retroviral fragments were identified in several bird species 32 .Among vultures, retroviral genome integration was previously documented solely in the turkey vulture genome, while avian endogenous retrovirus EAV-HP was reported in the facial skin microbiome of both black vultures and the turkey vultures 32,33 .Notably, this study marks the first identification of the retrovirusrelated sequences in the AKV genome.Furthermore, our analysis revealed the E3 ubiquitin-protein ligase ICP0 families, associated with the suppression of host defense responses.Within the ubiquitin proteasome system, E3 ubiquitin ligases play a crucial role in transferring ubiquitin to target substrates for subsequent proteolysis 34 .Specifically, ICP0 mediates the degradation of IRF7, a regulator of type I interferon production, which is a crucial cytokine for both innate and adaptive immunity 35 .
In terms of gene families or genes that were associated with the immune response and host defense mechanisms, certain highly enriched expanded gene families, such as CD163 and HFE, have been shown to be associated with immune responses and iron regulation.CD163 facilitates the endocytosis and clearance of hemoglobin/ haptoglobin complexes by macrophages, potentially protecting tissues from oxidative damage caused by free hemoglobin and serving as an innate immune sensor for microorganisms 36 .Additionally, HFE plays a significant role in the inflammatory response by modulating CD8 + T-lymphocyte responses due to its structural resemblance to MHC I 37 .In addition, HFE is implicated in iron regulation by inhibiting iron efflux from macrophages and enterocytes, potentially influencing susceptibility to bacterial infections due to iron's role as a nutrient for various pathogens 38 .

identified as an adhesion molecule present on T-cells, antigen-presenting cells and tumor cells according to KEGG pathways (map04514 and map05235
).It plays an important role in the PD1/ PD-L signaling pathway, regulating inappropriate immune responses 39 .AHR has been reported to activate Th17 differentiation in response to inflammatory stimuli 40 .Additionally, AHR is involved in xenobiotic metabolism by mediating CYP1A expression 41 .CYP1A in turn, plays pivotal roles in biotransformation of exogenous substances such as drugs and pollutants, as well as endogenous substances like hormones, vitamins and fatty acids 42 .MRC1, a glycoprotein located on the surface of antigen presenting cells, is crucial for cell-cell and pathogen recognition, mediating T-cell activation, antigen processing and presentation 43 .BIRC5, involved in positive regulation of the cell cycle response, is highly expressed in T cells and various tumor tissues 44,45 .Its high expression in tumors correlates positively with immune cell activation, suggesting its role as an immune-related gene 46 .Finally, SRM plays a pivotal role in spermidine biosynthesis, a natural polyamine with diverse functions including antioxidant properties, regulation of cell division, protein synthesis, tissue growth and reduction of lipid accumulation in adipose tissue 47,48 .However, the role of these gene families and genes in vultures should be further investigated.
In addition to host defense mechanisms, several genes or gene families with distinct yet related functions have been uncovered.These include species-specific gene families for feather keratin, as well as MYOF and ACTN1, which are highly enriched PSGs associated with muscle cell development.Beta keratin serves as a primary component of feather shafts, crucial for imparting stiffness and strength to withstand significant aerodynamic forces encountered during high-altitude soaring 49 .MYOF is expressed in both cardiac and skeletal muscle and plays a crucial role in muscle development by regulating the canonical Wnt signaling pathway 50,51 .Meanwhile, ACTN1 and ACTN4 are involved in actin filament bundling, cell adhesion and cell migration in non-muscle cells 52,53 .Apart from the AKV's gene families, prominent NWV-specific gene families have been linked to the sense of smell, including more than 10 olfactory receptor gene families.This finding aligns with previous studies, indicating that NWVs possess a heightened sense of smell attributed to a greater number of OR families compared to birds in the Accipitridae family 6 .
In terms of genetic relationships within the small population, SNPs located on putative Z and W chromosomes were excluded to mitigate potential bias stemming from sex chromosomes.This exclusion ensures a more accurate evaluation of genetic relatedness across the autosomal genome, allowing for a clearer understanding of population structure and genetic diversity.Despite the fact that these AKVs were donated at different time points and lack available pedigree or records, both phylogenetic tree analysis and MDS plot consistently revealed distinct subpopulations among the five AKV individuals.These subpopulation divisions were determined through genetic distance analyses based on identical by state (IBS) values.Upon the evaluation of inbreeding status, AKV4F and AKV5F exhibited a high proportion of ROH (F ROH ), indicating the possibility of parental consanguinity or close cousin mating for each animal.It is noteworthy that population history, particularly in species with a low effective population size, possibly influences ROH values and density 54 .From our data, it can be inferred that AKV4F and AVK5F likely originate from bottlenecked subpopulations that shared recent common ancestors.High genomewide homozygosity rates or high F ROH values are correlated with an increased risk of inbreeding depression, which has previously been associated with reproductive issues such as decreased fledgling production 55,56 .
Despite achieving success in breeding AKVs for the first time in 30 years, Thailand faces challenges as the remaining AKV breeders are aging, with a mean age of approximately 25.4 ± 3.6 years, and they lay eggs approximately once every 2 years.Furthermore, captive OWVs were reported to lay their first eggs at 7-9 years old, with the first offspring reproduced at 8-10 years of age 57 .This suggests that the repopulation of AKVs in Thailand may encounter a founder effect crisis in the next generation.To support the global conservation efforts of AKVs, it is crucial to investigate the genetic relatedness of captive AKVs worldwide, employing methods like affordable RADseq-based genome-wide SNPs, to facilitate breeding programs.Additionally, assisted reproductive technology (ART) techniques such as semen cryopreservation and artificial insemination should be integrated.This combined approach can help mitigate the adverse effects of closed genetic relatedness and contribute to the long-term health and sustainability of the population, setting a valuable model for conservation efforts in other endangered species.

Conclusion
Our study presents the first comprehensive genomic analysis of the AKV, shedding light on unique genetic features likely associated with their adaptation to scavenging carrion and soaring at high altitudes as well as potential mechanisms for modulating host defenses.Population genomics, utilizing genome-wide SNP data from RADseq, revealed that the AKV individuals were not genetically related to one another.However, two female AKVs showed a high risk of inbreeding.The utilization of genome-wide SNP data shows promise in guiding breeding programs for animals with limited pedigree information and assessing genetic relatedness before introducing individuals from different locations for breeding purposes.These insights significantly contribute to the conservation efforts of the AKV and hold potential relevance for other species facing similar challenges.

Ethical approval
All methods were performed in accordance with guidelines and regulations and the study was carried out in compliance with the ARRIVE guidelines.All animals were managed following the ethical guidelines required under the Chulalongkorn University Animal Care and Use Committee (CU-ACUC), Thailand (approval number 2131005).www.nature.com/scientificreports/

Positively selected genes
Coding sequences from 12 species in Accipitriformes and one species in Galliformes were utilized to analyze genes under positive selection using the branch-site model in CODEML implemented in PosiGene 63,64 .PSGs were identified based on criteria such as dN/dS > 1, P-value < 0.05 and FDR-adjusted P-value (Q-value) < 0.05.The significance of positive selection on these genes and the fold enrichment of specific biological processes and molecular functions associated with PSGs were analyzed using the DAVID database.In addition, the potential effects of amino acid replacements on protein functions were predicted using the Protein Variation Effect Analyzer (PROVEAN) software, compared with the red junglefowl genes 65 .The red junglefowl genome was selected as a reference because all PSGs of AKV were observed in the red junglefowl genome.GO enrichment analysis was also performed using the DAVID database.

Restriction-site associated DNA sequencing (RADseq)
Five AKV whole blood samples, comprising three males (AKV1M, AKV2M and AKV3M) and two females (AKV4F and AKV5F), were subjected to for genomic DNA extraction utilizing the Quick-DNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol.DNA concentrations were determined using both a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) and a Qubit fluorometer (Life Technologies).Assessment of sample integrity was conducted via pulsed-field gel electrophoresis.For library preparation, the DNA samples were diluted to a final concentration of 0.05 g/L.The RAD library was constructed following the manufacturer's instructions, employing the MGIEasy RAD Library Prep Kit (MGI Tech, Shenzhen, China).To achieve this, 1 µg of genomic DNA underwent enzymatic fragmentation using two restriction enzymes, TaqI and MseI.Subsequently, RAD adapters and barcodes were employed to ligate digested fragments.All resultant products from the various samples were pooled, and the quality of library construction was assessed using the Fragment Analyzer System (Agilent Technologies, Santa Clara, CA, USA).
The final PCR-amplified library fragments, ranging within the 400 − 450 bp, were transformed into a single-strand circularization DNA library and sequenced using the MGI Tech MGISEQ-2000RS platform, generating 150-bp paired-end reads.Raw reads were demultiplexed based on their barcodes, followed by the removal of sequences containing adaptors, barcodes, and low-quality reads from the dataset.

SNP calling and filtering
Raw reads obtained from RADseq were aligned to the reference genome, assembled in this study, utilizing Bowtie2 v2.3.5.1 66 .The resulting SAM files underwent conversion to BAM files using Samtools v1.10 67 .All BAM files were then utilized for SNP calling employing GATK v4.4.0.0.SNPs located on Z and W chromosomes were aligned against the Z and W chromosomes of the golden eagle (Accession No. GCF_900496995.4) using minimap2 68 .These SNPs were filtered out using vcftools (with parameters-remove-indels, -max-missing 1, minQ 30, minDP 3 and -not-chr for contigs likely containing sex-related sequences) and PLINK v1.9 (applying -maf 0.05, -geno 0, -mind 0 and -hwe 0.01) to eliminate low quality SNPs and mitigate potential biases arising from SNPs on putative sex chromosomes.Two SNP datasets were prepared: a non-LD-pruned dataset and an LD-pruned dataset.The latter was generated utilizing the parameters --indep-pairwise 50 5 0.2, specifying a 50-SNPs sliding window, a 5-SNP step size for shifting the window and an r 2 threshold of 0.2 for pruning.

Population structure and genetic relatedness
For the analysis of population structure, the LD-pruned dataset underwent analysis through phylogenetic tree construction and MDS plot generation.A phylogenetic tree was constructed using MEGA X software, employing the maximum likelihood method.The General Time Reversible model was determined to be the best fit for the analysis.The tree construction incorporated 1000 bootstraps to assess the robustness of the tree topology.MDS analysis was conducted using the -mds-plot command in PLINK v1.9.
For the analysis of IBD relatedness, the LD-pruned dataset was utilized to analyze pairwise IBD sharing, based on Pi-hat values.Additionally, probabilities of sharing zero (Z0), one (Z1) and two (Z2) IBD alleles were calculated using the --genome command in PLINK v1.9.The potential relatedness of each AKV was illustrated in a heatmap using R v4.1.1.For the inbreeding analysis, as there were insufficient LD-pruned SNPs for ROH analysis, the non-LD-pruned dataset was employed to detect ROH in each AKV individual.Sliding-window-based run detection was performed using the detectRUNs package version 0.9.6 within R v4.1.1 69 .Parameters were configured as follows: windowSize = 15, threshold = 0.05, minSNP = 20, maxOppWindow = 1, maxMissWindow = 1, maxGap = 10 6 , minLengthBps = 250,000, minDensity = 1/10 3 , maxOppRun = NULL and maxMissRun = NULL.The ROH segments were categorized by length into the following groups: 0-2 Mb, 2-4 Mb, 4-8 Mb, 8-16 Mb and > 16 Mb.Genome-wide F ROH values were estimated for each AKV.Individuals' F HOM were identified by analyzing observed homozygosity, expected homozygosity and non-missing genotypes using the --het command in PLINK.Additionally, a comparison between F ROH and F HOM was conducted by assessing the correlation between them using Pearson's correlation test.

Figure 1 .
Figure 1.KEGG pathway and Gene Ontology (GO) annotations of the Asian king vulture (AKV) genome.(a) Distribution of the number of proteins in different KEGG functional categories annotated using the KEGG Automatic Annotation Server (KAAS).(b) GO annotation of AKV.

Figure 2 .
Figure 2. Phylogenetic tree based on amino acid sequences derived from 1259 single-copy orthologous genes of Sarcogyps calvus and 11 avian species in the Order Accipitriformes, with red junglefowl (Gallus gallus) serving as the outgroup.(a) Chronogram.Estimated divergence times are indicated at each node.(b) Cladogram.The number at each node represents bootstrap values of 1000 replicates.Sarcogyps calvus is highlighted in red.

Figure 4 .
Figure 4. Phylogenetic tree showing gene family evolution.The number of expanded (green) and contracted (red) gene families is displayed at the nodes.The changes in gene family size were analyzed using CAFE software (v5) and visualized using the iTOL.

Figure 5 .
Figure 5. Population genomics of five Asian king vultures in Thailand.(a) Phylogenetic tree of captive AKV population.(b) Multidimensional scaling (MDS) plot of AKV population.(c) Heatmap of Pi-hat coefficient of AKV pairs.F, female; M, male.

Table 1 .
Comparison of genome assembly and quality assessment for Asian king vulture using PacBio and MGI hybrid sequencing with Purged analysis (PacBio-MGI-Purged) versus Nanopore and MGI hybrid sequencing (Nanopore-MGI).N/A, not applicable.

Table 2 .
Statistics of genetic diversity indices of AKV.F HOM , inbreeding coefficient from excess homozygosity.